Fig. S2.

ScPif1 repetitive translocation on (dT)₁₄₀. (A) A segment of the repetitive Cy3 enhancement (PIFE) signal observed when ATP is added to a surface immobilized 3′-dT-Cy3-(dT)₁₄₀ DNA previously incubated with 100 nM Pif1 and subsequently washed with buffer to remove unbound Pif1. The peak interval is defined as the time between the maximum of two Cy3 emission peaks. An example of a complete translocation event where the Pif1 reaches the 3′ end of the ssDNA is marked by c. The Pif1 then releases the ssDNA while remaining bound at the junction so that it can undergo another translocation event. An example of an incomplete translocation event where the Pif1 does not reach the 3′ end before releasing the ssDNA is marked by i. In the absence of ATP, no repetitive Cy3 enhancement is observed. (B) Histogram of peak intervals for Pif1 translocation at 50 μM ATP. The average peak interval was 2.0 ± 1.7 s and the median peak interval was 1.440 s (567 events collected from 54 molecules). (C) Histogram of peak intervals for Pif1 translocation at 5 mM ATP. The average peak interval was 1.1 ± 0.7 s and the median peak interval was 0.892 s (517 events collected from 33 molecules). Solutions conditions: 30 mM Tris⋅HCl, pH 8.1, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA, 0.5% (wt/vol) dextrose, 3 mM Trolox, 1 mg/mL glucose oxidase, and 0.4 mg/mL catalase, 25 °C.